CN106319055B - triple nucleic acid detection kit - Google Patents
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Abstract
The invention relates to a triple nucleic acid detection kit, which comprises a thallus lysate, a positive quantitative reaction liquid, a negative control reaction liquid and a thallus detection reaction liquid, wherein the thallus detection reaction liquid comprises specific primers of aspergillus, cryptococcus neoformans and candida albicans, a specific probe and an internal reference fluorescent dye, the three specific primers are designed according to areas of ITS sequences of the aspergillus, cryptococcus neoformans and candida albicans, marking report groups of the three specific probes respectively select FAM, VIC and CY-5, a quenching group is MGB, the excitation wavelength of the FAM group is 492nm, and the emission wavelength is 517 nm; excitation wavelength of VIC group is 557nm, emission wavelength is 574 nm; the emission wavelength of the CY-5 group is 646nm and the emission wavelength is 664 nm. The invention provides a triple nucleic acid quantitative detection kit for aspergillus, cryptococcus neoformans and candida albicans, which is used for assisting in diagnosing infection of the aspergillus, the cryptococcus neoformans and the candida albicans.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection of fluorescence quantitative PCR, in particular to a triple nucleic acid detection kit.
Background
at present, deep fungal infection is mostly caused by conditional pathogenic fungi, is mostly seen in people with low immunity, and has high disease death rate. Wherein, invasive fungal infection caused by candida accounts for 70-90% of all cases, and candida albicans accounts for 70-80% of candida infection; aspergillus infection accounts for 10% -20% of fungal infection; the cryptococcus is mainly cryptococcus neoformans, is a common pathogenic fungus for the co-infection of AIDS patients, causes cryptococcus meningitis of the AIDS patients, and has extremely low cure rate.
At present, 5 types of antifungal drugs are commonly used clinically, namely polyenes, azoles, echinocandins, fluorocytosines and allylamines. Among them, the most commonly used drugs mainly include fluconazole, itraconazole, 5-flucytosine, amphotericin B, nystatin, etc. Fluconazole and itraconazole are the first choice drugs for clinically treating deep fungal infection, have narrow antibacterial spectrum, are mainly used for treating candida and cryptococcus infections, have poor curative effect on aspergillus infections and the like, and are easy to generate drug resistance. In addition, the medicines used for candida albicans, aspergillus and cryptococcus neoformans are different in clinical preventive medication, empirical treatment, preemptive treatment and target treatment of four medication modes. And still, due to the lack of definite diagnosis basis at present, the antibiotic abuse still exists, however, the drug resistance of the patient caused by the antibiotic abuse even leads to the patient to be non-drug-salvageable, so that a rapid and accurate detection method is needed.
At present, the fungus detection methods include, in addition to the culture method, a latex agglutination test, a colloidal gold method, and an Elisa test and a G test. The culture method is gold standard, but it takes long time and has low positive rate. The latex agglutination test and the colloidal gold method have high sensitivity, but the false positive is high, the latex titer cannot accurately reflect the infection condition, the G test only detects fungi and cannot distinguish different strains, the Elisa test easily has a false positive result, and the operation is complex. The fluorescent quantitative PCR method can directly reflect the infection condition of a patient through the quantification, can perform periodic detection to achieve the effect of guiding prognosis, and has a marking meaning for clinical diagnosis.
The method of fluorescence quantitative PCR is highly advantageous in sensitivity, specificity and operation, so that the method has great significance for early treatment of fungal infection when being applied to clinical fungal detection. The Q-PCR technology is applied to the field of fungus detection, fills the domestic blank, realizes early, rapid and accurate detection, and provides a new way for detecting fungi for clinic. The detection of three fungi, namely candida albicans, aspergillus and cryptococcus neoformans, is completed simultaneously in one detection, and the clinical diagnosis is assisted more comprehensively. The detection kit is matched with a portable Q-PCR instrument for detection, complicated setting and running procedures are not needed, only detection items and calibrated sample names are needed to be selected, after the reaction is finished, background software analyzes data in a targeted manner, a detection report is directly given, and the detection kit is convenient and rapid.
The currently published patents CN201110214986.9 and CN201410821483.1 relate to universal primers, detection probes and kits for detecting pathogenic aspergillus, and mainly aim at detecting four pathogenic aspergillus including aspergillus fumigatus, aspergillus flavus, aspergillus terreus and aspergillus niger; both patents CN201110417477.6 and CN201410017428.7 are PCR fluorescence detection kits for detecting candida albicans; CN201410017251.0 this patent relates to a nucleic acid detection kit for detecting a cryptococcus neoformans. The above patents all describe methods for detecting a single species, and do not achieve the simultaneous detection of multiple fungi like the present invention.
CN201410820825.8 this patent relates to a method for detecting common pathogenic fungi, which mainly comprises three primers forming two pairs of primers, two probes forming a universal test, the positive result of which represents single or mixed infection of 5 species of aspergillus, cryptococcus, candida, rhizopus oryzae and mucor circinelloides, the method is directed to the detection of fungi of large class, and does not have the ability of identifying which species of fungi is specific like the present invention.
In addition, the patent CN201510920311.4 relates to a nucleic acid fluorescence qualitative detection kit for enterovirus universal, coxsackievirus a16 type and enterovirus 71 type, and the patent jointly detects three viruses, but is qualitative detection and aims at virus detection, the difference of the designed position sequence of a primer is large, the design of the primer is facilitated, and is not at a difficulty level with the invention. Therefore, no relevant product exists at present.
In various products of joint inspection, due to the similarity of gene sequences, the mutual interference condition as shown in fig. 1a is very likely to occur, so that the accuracy of the experimental result is reduced, the specificity is reduced, and the clinical assistance cannot be realized. Therefore, the technical difficulty must be overcome, and the experimental team solves the problem by multiple searching and verification experiments aiming at the sequence and the fluorescent group of the probe, and as shown in fig. 2a, the mutual interference does not exist.
by contrast, the above disclosed patent technical solutions and the quantitative detection scheme of the present application, in which three common pathogenic bacteria are mixed in one system for simultaneous detection, and the species categories can be distinguished, have substantial differences.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a triple nucleic acid detection kit.
The technical scheme for realizing the purpose of the invention is as follows:
A triple nucleic acid detection kit comprises a thallus lysate, a positive quantitative reaction liquid, a negative control reaction liquid and a thallus detection reaction liquid, wherein the thallus detection reaction liquid comprises specific primers, specific probes and internal reference fluorescent dyes of aspergillus, cryptococcus neoformans and candida albicans,
Wherein three specific primers in the thallus detection reaction liquid are designed according to the areas of the ITS sequences of aspergillus, cryptococcus neoformans and candida albicans,
Wherein, the labeled reporter groups of the three specific probes in the thallus detection reaction liquid are respectively selected from FAM, VIC and CY-5, the quenching group is MGB, the excitation wavelength of the FAM group is 492nm, and the emission wavelength is 517 nm; excitation wavelength of VIC group is 557nm, emission wavelength is 574 nm; the emission wavelength of the CY-5 group is 646nm, and the emission wavelength is 664 nm; the excitation wavelengths and the emission wavelengths of the FAM, VIC and CY-5 groups do not have cross overlapping sections, so that the mutual interference phenomenon does not exist, and the simultaneous specificity detection of the three bacteria is realized;
Wherein the components of the thallus detection reaction liquid are as follows:
And the specific primer and the specific probe sequence in the thallus detection reaction liquid are respectively as follows:
Aspergillus specific primer forward: SQ 15 '-AAGCACGGCTTGTGTGTTGG-3'
Reverse direction of the aspergillus specific primer: SQ 25 '-AGCCCCATACGCTCGAGGA-3'
Aspergillus-specific probes: SQ 35 '-FAM-AAGGCAGCGGCGGCA-MGB-3'
Cryptococcus neoformans specific primers forward: SQ 45 '-GTTTTATTACCTGTTGGACTTGGATTTG-3'
The cryptococcus neoformans specific primer is reversed: SQ 55 '-GGTTGTTATCAGCAAGCCGAAG-3'
Cryptococcus typhae specific probe: SQ 65 '-VIC-TGTGTTAGTGGGAAGGT-MGB-3'
Candida albicans specific primer forward: SQ 75 '-CGCTGGGTTTGGTGTTGAG-3'
Candida albicans specific primer reverse: SQ 85 '-CGCCTTACCACTACCGTCTTTC-3'
candida albicans specific probe: SQ 95 '-CY 5-AATACGACTTGGGTTTGCT-MGB-3';
Furthermore, the reference fluorescent dye was RQX, and the homogenization corrected for differences in volume in the batch due to pipetting errors or evaporation, and corrected for signal changes due to foaming or evaporation, condensation in a single reaction run.
the invention has the advantages and effects that:
1. the invention carries out combined detection aiming at three pathogenic fungi, and is quantitative detection, the 18S, ITS conserved sequence of the invention has higher similarity among different strains, and the design difficulty of the primer is higher.
2. The invention provides a triple nucleic acid quantitative detection kit for aspergillus, cryptococcus neoformans and candida albicans, which adopts polymerase chain reaction and fluorescent probe technology to respectively carry out amplification detection on sequences with high conservative specificity of the aspergillus, the cryptococcus neoformans and the candida albicans, and assists in diagnosing infection of the aspergillus, the cryptococcus neoformans and the candida albicans.
Drawings
FIG. 1 is a graph of a set of amplification curves for interactions where there is a crossover between probe wavelengths, where FIG. 1a is a graph of three amplification curves for a crossover; FIG. 1b is a graph of normal amplification of Candida; FIG. 1c is a graph of Aspergillus uncrossed amplification; FIG. 1d is a graph of latent sphere amplification resulting in crossover;
FIG. 2 is a graph of a set of amplification curves for the absence of cross-over between probe wavelengths without interaction, wherein FIG. 2a is a graph of three amplification curves without cross-over; FIG. 2b is a graph of normal amplification of Candida; FIG. 2c is a graph of Aspergillus uncrossed amplification; FIG. 2d is a graph of amplification without crypt crossing.
Detailed Description
the present invention will now be further described with reference to specific embodiments, which are to be construed as merely illustrative, and not restrictive, of the scope of the invention, as defined in the following examples.
A triple nucleic acid detection kit comprises a thallus lysate, a positive quantitative reaction liquid, a negative control reaction liquid and a thallus detection reaction liquid, wherein the thallus detection reaction liquid comprises specific primers, specific probes and internal reference fluorescent dyes of aspergillus, cryptococcus neoformans and candida albicans,
Wherein, three specific primers in the thallus detection reaction liquid are designed according to the areas of the ITS sequences of aspergillus, cryptococcus neoformans and candida albicans.
wherein, the labeled reporter groups of the three specific probes in the thallus detection reaction liquid are respectively selected from FAM, VIC and CY-5, the quenching group is MGB, the excitation wavelength of the FAM group is 492nm, and the emission wavelength is 517 nm; excitation wavelength of VIC group is 557nm, emission wavelength is 574 nm; the emission wavelength of the CY-5 group is 646nm, and the emission wavelength is 664 nm; the excitation wavelengths and the emission wavelengths of the FAM, VIC and CY-5 groups do not have cross overlapping sections, so that the mutual interference phenomenon does not exist, and the simultaneous specificity detection of the three bacteria is realized;
Wherein the components of the thallus detection reaction liquid are as follows:
In the specific implementation of the invention, the sequences of the specific primer and the specific probe in the thallus detection reaction solution are respectively as follows:
Aspergillus specific primer forward: SQ 15 '-AAGCACGGCTTGTGTGTTGG-3'
Reverse direction of the aspergillus specific primer: SQ 25 '-AGCCCCATACGCTCGAGGA-3'
Aspergillus-specific probes: SQ 35 '-FAM-AAGGCAGCGGCGGCA-MGB-3'
Cryptococcus neoformans specific primers forward: SQ 45 '-GTTTTATTACCTGTTGGACTTGGATTTG-3'
the cryptococcus neoformans specific primer is reversed: SQ 55 '-GGTTGTTATCAGCAAGCCGAAG-3'
Cryptococcus typhae specific probe: SQ 65 '-VIC-TGTGTTAGTGGGAAGGT-MGB-3'
Candida albicans specific primer forward: SQ 75 '-CGCTGGGTTTGGTGTTGAG-3'
Candida albicans specific primer reverse: SQ 85 '-CGCCTTACCACTACCGTCTTTC-3'
candida albicans specific probe: SQ 95 '-CY 5-AATACGACTTGGGTTTGCT-MGB-3';
In a specific implementation of the invention, the reference fluorescent dye is RQX, and the homogenization corrects for volume differences in the batch due to pipetting errors or evaporation, and corrects for signal changes due to foaming or evaporation, condensation in a single reaction run.
example (c): preparation of the kit
(1) Primer and probe design and synthesis
The specific primer sequences are respectively high-conservative and strong-specificity sequences aiming at ITS of aspergillus, cryptococcus neoformans and candida albicans, the labeled reporter groups of the three specific probes are also respectively selected FAM, VIC and CY-5, the quenching group is MGB, the data of the three specific probes are all derived from ribosome ITS sequences in GenBank, and the primers and the probes are synthesized by life technologies.
(2) Preparation of positive quantitative reference substance and negative reference substance
respectively taking the extracted aspergillus genome, cryptococcus neoformans genome and candida albicans genome as templates, amplifying by using aspergillus primers, dNTPs, dUTP, 10 Taq buffer and Taq enzyme as reaction systems to obtain an aspergillus positive reference substance, amplifying by using the cryptococcus neoformans primers, dNTPs, dUTP, 10 Taq buffer and Taq enzyme as reaction systems to obtain a cryptococcus neoformans positive reference substance, and amplifying by using the candida albicans primers, dNTPs, dUTP, 10 Taq buffer and Taq enzyme as reaction systems to obtain the candida albicans positive reference substance. And then purifying the PCR product by using a PCR purification kit, measuring the OD value of each positive reference substance by using a spectrophotometer, and obtaining the concentration by using a copy number conversion formula. And sequentially diluted to 5X 107copies～5×102And (3) adding the copies with five gradients into the positive reaction solution respectively to prepare a positive quantitative standard substance, and adding the clinical negative sample for negative control into the negative reaction solution after the clinical negative sample is treated by the lysis solution to prepare a negative control substance.
(3) reaction solution composition of thallus detection reaction solution
Use of the kit
Sample processing
(1) Alveolar lavage fluid specimen treatment
mixing the alveolar lavage fluid in the tube by a pipette, taking out 1000 microliter of the alveolar lavage fluid in a new centrifuge tube, centrifuging for 5min at 12,000rpm (about 13800g), and carefully discarding the supernatant (the centrifuge tube can be inverted on clean paper to remove the liquid at the tube opening);
② adding 100 mul of thallus lysate (immediately taking out the thallus lysate after fully mixing the thallus lysate before adding liquid), fully mixing, carrying out water bath at 100 ℃ for 10min, and centrifuging at 10,000rpm for 2min for standby.
(2) Enzyme treatment: the DNA polymerase and UNG enzyme were centrifuged at 8,000rpm for 30s before use. If positive quantitative reaction liquid is prepared, 32 mul of diluent is added into a tube of DNA polymerase, and the mixture is fully and evenly mixed until the mixture is added into the positive quantitative reaction liquid. If thallus detection reaction and negative reaction liquid are prepared, firstly 8 mul UNG enzyme is added into a tube of DNA polymerase, then 24 mul diluent is added, and the mixture is fully and uniformly mixed and added into the thallus detection reaction liquid and the negative reaction liquid.
(3) Sample detection
Mu.l of the treated sample supernatant was added to the cell detection reaction tube, and 2. mu.l of the enzyme prepared previously was added to the cell detection reaction tube. For a control test, 2. mu.l of diluted DNA polymerase (without UNG enzyme) was added to the reaction solution for negative detection and the reaction solution for positive quantitative detection, respectively.
(4) reaction procedure
A report group of aspergillus of the probe is set as FAM, cryptococcus neoformans is set as VIC, candida albicans is set as CY-5, and quenching groups are set as MGB. Opening a parameter window setting loop:
10 minutes at 37 DEG C
the temperature of the mixture is 95 ℃ for 5 minutes,
95 ℃ for 15 seconds → 62 ℃ for 30 seconds → 40 cycles
(5) Result judgment
And adjusting the initial value, the stop value and the threshold value of the baseline parameter according to the analyzed image (the user can adjust the initial value by himself according to the actual situation, the initial value can be 1-10, and the stop value can be selected in the range of 5-20), so that the standard curve graph under the standard curve window is optimal. And analyzing the result under a quantitative analysis menu, and determining the concentration of the sample according to the position of the sample point on the standard curve.
If the growth curve does not show S-shaped curve or Ct value > is 35, judging that the total content of the sample is less than the detection limit.
If the growth curve is S-shaped and Ct value is less than 35, judging according to the following method:
If the Ct value of the sample is less than 103Positive quantitative reference substance, the total DNA content of the sample is less than 1.0 multiplied by 103Copying the gene;
If the Ct value of the sample is 103to 107And (3) among positive quantitative reference products, determining that the total DNA content of the sample is C gene copy;
If the C of the sample is greater than 107Positive quantitative reference substance, the total DNA content of the sample is>1.0×107And (4) copying the gene. If accurate quantification is required, the extracted sample can be diluted to a linear range for detection.
Claims (2)
1. A triple nucleic acid detection kit comprises a thallus lysate, a positive quantitative reaction solution, a negative control reaction solution and a thallus detection reaction solution, and is characterized in that: the thallus detection reaction liquid contains specific primers of aspergillus, cryptococcus neoformans and candida albicans, specific probes and internal reference fluorescent dyes, wherein the three specific primers in the thallus detection reaction liquid are designed according to areas of ITS sequences of the aspergillus, cryptococcus neoformans and candida albicans, marking report groups of the three specific probes in the thallus detection reaction liquid are respectively FAM, VIC and CY-5, a quenching group is MGB, the excitation wavelength of the FAM group is 492nm, and the emission wavelength is 517 nm; excitation wavelength of VIC group is 557nm, emission wavelength is 574 nm; the emission wavelength of the CY-5 group is 646nm, and the emission wavelength is 664 nm; the excitation wavelengths and the emission wavelengths of the FAM, VIC and CY-5 groups do not have cross overlapping sections, so that the mutual interference phenomenon does not exist, and the simultaneous specificity detection of the three bacteria is realized;
the components of the thallus detection reaction liquid are as follows:
The specific primer and the specific probe sequence in the thallus detection reaction liquid are respectively as follows:
Aspergillus specific primer forward: SQ 15 '-AAGCACGGCTTGTGTGTTGG-3'
Reverse direction of the aspergillus specific primer: SQ 25 '-AGCCCCATACGCTCGAGGA-3'
Aspergillus-specific probes: SQ 35 '-FAM-AAGGCAGCGGCGGCA-MGB-3'
Cryptococcus neoformans specific primers forward: SQ 45 '-GTTTTATTACCTGTTGGACTTGGATTTG-3'
The cryptococcus neoformans specific primer is reversed: SQ 55 '-GGTTGTTATCAGCAAGCCGAAG-3'
cryptococcus typhae specific probe: SQ 65 '-VIC-TGTGTTAGTGGGAAGGT-MGB-3'
Candida albicans specific primer forward: SQ 75 '-CGCTGGGTTTGGTGTTGAG-3'
candida albicans specific primer reverse: SQ 85 '-CGCCTTACCACTACCGTCTTTC-3'
Candida albicans specific probe: SQ 95 '-CY 5-AATACGACTTGGGTTTGCT-MGB-3'.
2. The triple nucleic acid detection kit according to claim 1, characterized in that: the internal reference fluorescent dye is RQX.
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Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101038254A (en) * | 2007-04-29 | 2007-09-19 | 南方医科大学 | PCR kit for fluorescence quantitative detecting aspergilli |
| CN101555522A (en) * | 2009-05-14 | 2009-10-14 | 浙江大学 | Kit used for detecting candida albicans in intestinal tract by fluorescence quantitative PCR method |
| CN102719529A (en) * | 2012-05-04 | 2012-10-10 | 辽宁科骏生物有限公司 | Trichomonas vaginalis and candida albicans two-channel fluorescence PCR detection method and kit thereof |
| CN102876585A (en) * | 2012-09-19 | 2013-01-16 | 中国人民解放军第二军医大学 | Cryptococcus gattii, and isolated culture method and application thereof |
| CN103740832A (en) * | 2014-01-15 | 2014-04-23 | 湖南圣维尔医学检验所有限公司 | Cryptococcus neoformans detecting kit |
| CN103740834A (en) * | 2014-01-15 | 2014-04-23 | 湖南圣维尔医学检验所有限公司 | Candida albicans detection kit |
| CN103882135A (en) * | 2014-03-31 | 2014-06-25 | 深圳意达凯生物科技有限公司 | Primer pair for detecting candida albicans, kit and application thereof |
| CN104164511A (en) * | 2014-08-25 | 2014-11-26 | 天津喜诺生物医药有限公司 | Aspergillus quantitative detection fluorescence PCR (polymerase chain reaction) kit |
| CN104450936A (en) * | 2014-12-25 | 2015-03-25 | 天津宝瑞生物技术有限公司 | Fluorescence quantitative PCR primer, probe and kit for detecting ordinary pathogenic fungi |
| CN104531897A (en) * | 2014-12-05 | 2015-04-22 | 广东省农业科学院动物卫生研究所 | Method and kit for three-color fluorescent PCR detection of Classical swine feine virus (CSFV), porcine reproductive and respiratory syndrome virus-U (PRRSV-U) and porcine reproductive and respiratory syndrome virus-M (PRRSV-M) |
| CN104762392A (en) * | 2015-04-09 | 2015-07-08 | 山东省动物疫病预防与控制中心 | Triple fluorogenic quantitative PCR detection method of escherichia coli, Klebsiella pneumoniae and acinetobacter |
| CN105274199A (en) * | 2015-06-17 | 2016-01-27 | 厦门医学高等专科学校 | A reagent kit simultaneously detecting Staphylococcus aureus and Cronobacter sakazakii, and usage method thereof |
-
2016
- 2016-08-30 CN CN201610765955.5A patent/CN106319055B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101038254A (en) * | 2007-04-29 | 2007-09-19 | 南方医科大学 | PCR kit for fluorescence quantitative detecting aspergilli |
| CN101555522A (en) * | 2009-05-14 | 2009-10-14 | 浙江大学 | Kit used for detecting candida albicans in intestinal tract by fluorescence quantitative PCR method |
| CN102719529A (en) * | 2012-05-04 | 2012-10-10 | 辽宁科骏生物有限公司 | Trichomonas vaginalis and candida albicans two-channel fluorescence PCR detection method and kit thereof |
| CN102876585A (en) * | 2012-09-19 | 2013-01-16 | 中国人民解放军第二军医大学 | Cryptococcus gattii, and isolated culture method and application thereof |
| CN103740832A (en) * | 2014-01-15 | 2014-04-23 | 湖南圣维尔医学检验所有限公司 | Cryptococcus neoformans detecting kit |
| CN103740834A (en) * | 2014-01-15 | 2014-04-23 | 湖南圣维尔医学检验所有限公司 | Candida albicans detection kit |
| CN103882135A (en) * | 2014-03-31 | 2014-06-25 | 深圳意达凯生物科技有限公司 | Primer pair for detecting candida albicans, kit and application thereof |
| CN104164511A (en) * | 2014-08-25 | 2014-11-26 | 天津喜诺生物医药有限公司 | Aspergillus quantitative detection fluorescence PCR (polymerase chain reaction) kit |
| CN104531897A (en) * | 2014-12-05 | 2015-04-22 | 广东省农业科学院动物卫生研究所 | Method and kit for three-color fluorescent PCR detection of Classical swine feine virus (CSFV), porcine reproductive and respiratory syndrome virus-U (PRRSV-U) and porcine reproductive and respiratory syndrome virus-M (PRRSV-M) |
| CN104450936A (en) * | 2014-12-25 | 2015-03-25 | 天津宝瑞生物技术有限公司 | Fluorescence quantitative PCR primer, probe and kit for detecting ordinary pathogenic fungi |
| CN104762392A (en) * | 2015-04-09 | 2015-07-08 | 山东省动物疫病预防与控制中心 | Triple fluorogenic quantitative PCR detection method of escherichia coli, Klebsiella pneumoniae and acinetobacter |
| CN105274199A (en) * | 2015-06-17 | 2016-01-27 | 厦门医学高等专科学校 | A reagent kit simultaneously detecting Staphylococcus aureus and Cronobacter sakazakii, and usage method thereof |
Non-Patent Citations (2)
| Title |
|---|
| PCR-RFLP鉴别念珠菌、曲霉和隐球菌的探讨;秦振宇等;《临床皮肤科杂志》;20000430;第29卷(第2期);76-78 * |
| 我国真菌感染的实验室检测现状;章强强;《诊断学理论与实践》;20160225;第15卷(第1期);1-4 * |
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Effective date of registration: 20191210 Address after: 19 Jingwu Road, Beihai Industrial Park, Guangxi Zhuang Autonomous Region Patentee after: Beihai Xinglong biological product Co., Ltd. Address before: 300480, No. 8 building, foreshore Industry Park, Binhai New Area, Tianjin, No. 2, No. two, waterfront industrial park Patentee before: TIANJIN XINUO BIOLOGICAL PHARMACEUTICAL CO., LTD. |